Phage integrases are enzymes that mediate site-specific recombination between two DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB. Typically, integrases are grouped into two major families based on their mode of catalysis. The tyrosine family integrases utilize a catalytic tyrosine to mediate strand cleavage, and some of them require accessory protein(s) for the reaction. The serine family integrases use a catalytic serine for strand cleavage.
Phage integrases mediate efficient site-specific recombination between two different sequences that are relatively short, enabling them to manipulate eukaryotic cells with large genomes, such as mammals.
The Integrase (Int) recombinase of the coliphage HK022 mediates integration and excision of the bacteriophage DNA into and out of a specific chromosomal site on its Escherichia coli host. These two site-specific recombination reactions occur between two defined pairs of DNA attachment (att) sites. The bacterial attB consists of a 21 base pair (bp) core sequence (BOB′) that is composed of 7 central bps, defined as the overlap O (the site of DNA exchange), flanked by two 7 bp inverted repeats (B and B′) that serve as binding sites for Int. The corresponding site on the phage chromosome attP is composed of a similar core (COC′) that is flanked by two long arms (P and P′ of 157 and 85 bps, respectively) carrying additional binding sites for Int and for the accessory DNA bending proteins (IHF, and Xis) that, in E. coli, are required for the reaction. When bivalent Int monomers and accessory proteins are bound to the P and P′ arms of attP, the latter's bend the arms, thereby facilitating binding of the catalytic domain of Int to the core, the site of the reaction. Subsequently, this proteo-DNA complex (the intasome) captures attB to perform the recombination reaction. Phage excision occurs between the recombinant attL (BOP′) and attR (POB′) sites that flank the integrated prophage, each composed of the recombined core and one of the arms. Since the integration/excision reactions use a different pair of att sites and a different composition of the accessory proteins, they are not completely reversible (Weisberg et al., 1999; Azaro and Landy, 2002).
The wild type Int of HK022 was developed by some of the inventors of the present invention, to catalyze site-specific integration and excision of plasmid DNA into the human genome in cell cultures, as well as in Arabidopsis plants and Anabaena algae when supplied with the relevant att sites (Harel-Levy G. et al., 2008; Gottfried et al., 2005; Melnikov et al., 2009). In the eukaria, Int is active in integrative as well as in excisive reactions without the need to supply any of the accessory proteins (i.e. IHF and Xis), that are required for these reactions in E. coli. Nevertheless, it has been demonstrated by some of the inventors of the present invention that an int gene that was modified for optimal human codon usage and the presence of the accessory proteins significantly improved the recombination activities of Int in the human cells (Malchin et al., 2009).
U.S. Pat. No. 7,732,585 discloses a nucleic acid encoding an altered unidirectional site-specific bacteriophage integrase that has integrase activity, wherein said altered integrase has improved recombination efficiency towards wild-type or pseudo attachment sites as compared to a corresponding wild-type integrase.
U.S. Pat. No. 7,670,823 discloses an isolated nucleic acid molecule comprising a lambda att recombination site wherein a sequence of the seven base pair overlap region within the fifteen base pair core region is ATTATAC. It is further disclosed that the lambda att-recombination site is located between a transcriptional regulatory sequence and an open reading frame, wherein the transcriptional regulatory sequence and open reading frame are operably linked.
PCT Patent Application Publication No. WO2001/007572 discloses methods for obtaining integration of nucleic acids into eukaryotic cells. The publication discloses site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the ΦC31 integrase, that can mediate recombination between the recombination sites, but not between hybrid recombination sites that are formed upon the recombination.
PCT Patent Application Publication No. WO2001/038511 discloses a method for the sequence-specific recombination of DNA in eukaryotic cells, comprising: introducing a first DNA sequence which contains a res sequence and copy of the first DNA sequence into a cell, and performing the sequence-specific recombination using the influence of a resolvase.
U.S. Patent Application Publication No. 2003/027337 discloses a method of sequence specific recombination of DNA in eukaryotic cells utilizing att sequences from the bacteriophage lambda. The publication further discloses a method comprising performing the sequence specific recombination of DNA with an Int and a Xis factor.
There remains an unmet need for compositions and methods enabling specific and efficient expression of exogenous genes of interest specifically in target cells and not in other cell populations.